Para-hydroxycinnamic acid (pHCA) is a high-value, aromatic chemical compound that may be used as a monomer for the production of Liquid Crystal Polymers (LCP). LCPs are polymers that exhibit an intermediate or mesophase between the glass-transition temperature and the transition temperature to the isotropic liquid or have at least one mesophase for certain ranges of concentration and temperature. The molecules in these mesophases behave like liquids and flow, but also exhibit the anisotropic properties of crystals. LCPs are used in liquid crystal displays, and in high-speed connectors and flexible circuits for electronic, telecommunication, and aerospace applications. Because of their resistance to sterilizing radiation and their high oxygen and water vapor barrier properties, LCPs are also used in medical devices, and in chemical and food packaging.
Due to its importance as a high-value, aromatic chemical compound, chemical synthesis of pHCA is known. However, chemical synthesis is expensive due to the high energy needed for synthesis and the extensive product purification required. Biological production of pHCA offers a low cost, simplified solution to the problem.
The production of pHCA by plants using the enzymes phenylalanine ammonia lyase (PAL) (EC 4.3.1.5) and a P450 enzyme is well known. Phenylalanine ammonia-lyase is widely distributed in plants (Koukol et al., J. Biol. Chem. 236:2692–2698 (1961)), fungi (Bandoni et al., Phytochemistry 7:205–207 (1968)), yeast (Ogata et al., Agric. Biol. Chem. 31:200–206 (1967)), and Streptomyces (Emes et al., Can. J. Microbiology 48:613–622 (1970)), but it has not been found in Escherichia coli or mammalian cells (Hanson and Havir In The Enzymes, 3rd ed.; Boyer, P., Ed.; Academic: New York, 1967; pp 75–167). PAL is the first enzyme of phenylpropanoid metabolism and catalyzes the removal of the (pro-3S)-hydrogen and —NH3+ from L-phenylalanine to form trans-cinnamic acid. In the presence of a P450 enzyme system, trans-cinnamic acid can be converted to para-hydroxycinnamic acid (pHCA) which serves as the common intermediate in plants for production of various secondary metabolites such as lignin and isoflavonoids. In microbes however, cinnamic acid and not pHCA acts as the precursor for secondary metabolite formation. No cinnamate hydroxylase enzyme has so far been characterized from microbial sources. The PAL enzyme in plants is thought to be a regulatory enzyme in the biosynthesis of lignin, isoflavonoids and other phenylpropanoids (Hahlbrock et al., Annu. Rev. Plant Phys. Plant Mol. Biol. 40:347–369 (1989)). However, in the red yeast, Rhodotorula glutinis (Rhodosporidium toruloides), this lyase degrades phenylalanine as a catabolic function and the cinnamate formed by the action of this enzyme is converted to benzoate and other cellular materials.
Genes encoding PAL are known in the art and several have been sequenced from both plant and microbial sources (see for example EP 321488 [Rhodosporidium toruloides]; WO 9811205 [Eucalyptus grandis and Pinus radiata]; WO 9732023 [Petunia]; JP 05153978 [Pisum sativum]; WO 9307279 [potato, rice]; and for example GenBank AJ010143 and X75967). The PAL genes from various sources have been over-expressed as active PAL enzymes in yeast, Escherichia coli and insect cell cultures (Faulkner et al., Gene 143:13–20 (1994); Langer et al., Biochemistry 36:10867–10871 (1997); McKegney et al., Phytochemistry 41:1259–1263 (1996)).
Some PAL genes, in addition to their ability to convert phenylalanine to cinnamate, can accept tyrosine as substrate. In such reactions the enzyme activity is designated tyrosine ammonia lyase (TAL). Conversion of tyrosine by TAL results in the direct formation of pHCA from tyrosine without the intermediacy of cinnamate. However, there has been only one, very recent report of a gene which encodes an enzyme having significantly higher TAL catalytic activity than PAL activity (Kyndt et al., FEBS Letters 512:240–244 (2002)). This gene was isolated from the bacterium Rhodobacter capsulatus and encoded an enzyme that had a TAL catalytic efficiency that was approximately 150 times higher than that for PAL. This TAL protein was reported to have a higher homology to the PAL proteins of plants (e.g., 32% identity with the PAL sequence of Pinus taeda), than to the PAL sequences of yeasts. All other natural PAL/TAL enzymes prefer to use phenylalanine rather than tyrosine as their substrate. The wild-type PAL/TAL enzyme from the yeast Rhodosporidium exhibits a reduced preference for phenylalanine as compared to tyrosine, having a ratio of TAL catalytic activity to PAL catalytic activity of only 0.58 (reported in Hanson and Havir, In The Biochemistry of Plants; Academic: New York, 1981; Vol. 7, pp 577–625). For comparison, the PAL/TAL enzymes studied in other organisms typically possess PAL/TAL ratios of 15 or greater. Sariaslani et al. (U.S. Patent Application No. 60/383232) describe an inducible TAL enzyme that was isolated from the yeast Trichosporon cutaneum. This enzyme had a higher TAL than PAL activity with a PAL/TAL activity ratio of 0.73.
U.S. Pat. No. 6,368,837 discloses several methods for the biological production of pHCA. These include: the incorporation of the wild type PAL from the yeast Rhodotorula glutinis into E. coli and utilizing the ability of the wild type PAL to convert tyrosine to pHCA; the incorporation of the wild type PAL from the yeast Rhodotorula glutinis plus the plant cytochrome P-450 and the P-450 reductase into E. coli to convert phenylalanine to cinnamic acid and then to pHCA; and the development of a mutant PAL/TAL gene that encoded an enzyme with enhanced TAL activity. This mutant gene was isolated by mutagenesis of the wild type Rhodosporidium toruloides PAL and encoded an enzyme with a TAL/PAL ratio of 1.7. This gene was used to produce PCHA by direct conversion of tyrosine. The development of several other mutant PAL/TAL genes that encode enzymes with enhanced TAL activity is disclosed by Tang in U.S. Pat. No. 6,521,748. TAL/PAL ratios up to 7.2 were reported from these mutant genes. However, other enzymes with higher TAL activity are required for the economical production of PCHA.
The problem to be solved therefore is to obtain a naturally occurring enzyme with higher TAL than PAL activity to be used for the direct conversion of tyrosine to pHCA and to serve as a tool for future enzyme engineering to produce more efficient TAL enzymes. Applicants have solved the stated problem by isolating an enzyme from the bacterium Rhodobacter sphaeroides that has a higher TAL catalytic activity than PAL activity.